The ability of any pathogenic bacterium to persist in the host depends on the expression of factors required for colonization, evasion of host defenses, and for acquisition of nutrients. All organisms require iron, and bacteria unable to assimilate this important nutrient demonstrate reduced virulence. In response to iron limitation many organisms express elaborate iron acquisition systems; some pathogenic bacteria also exhibit an increased expression of toxins under iron stress. The overall goals of this research are to analyze two reported iron acquisition systems in Bordetella pertussis. Experiments in this proposal are designed to ascertain if the two systems are distinct or if there exist any common molecular aspects. System 1, proposed to be mediated by a cell surface receptor which directly removes iron from transferrin and lactoferrin, will be characterized by 55Fe-transferrin and 55Fe-lactoferrin transport experiments. The receptor(s) will be identified and purified on the basis of its affinity for ligand. Antibodies specific for the receptors will be used to inhibit transferrin and lactoferrin-mediated iron transport and to screen System I mutants. System 11 is thought to function through a soluble iron-binding siderophore. The siderophore will be purified, characterized, and its ability to supply iron to B. pertussis evaluated in 55Fe-siderophore uptake experiments. Transposon insertion mutations (using Tn5 lac and TnphoA) will be generated to identify genes which will distinguish specific functions of each iron system. After identification of genes involved in iron acquisition by mutational analysis, the wild-type genes will be isolated from a B. pertussis gene library and their nucleotide sequences determined, Selected mutants deficient in System I or System II cell surface receptors, other periplasmic or inner membrane transport functions, siderophore biosynthesis, or regulatory function will be evaluated for production of virulence-associated determinants including pertussis toxin, dermonecrotic toxin, and adenylate cyclase/hemolysin. Infection experiments using the mutants in a mouse model system will determine the contribution of the iron systems to pathogenesis. This research is important to establish the means by which B. pertussis obtains iron in the growth-limiting environment of the host and to determine the effects of iron stress on the expression of virulence factors. Because it has been conclusively shown that iron is important to bacterial pathogenesis and that hosts mount immune responses to components of iron acquisition systems, this research has great potential for vaccine application in addition to yielding important basic physiological information about B. pertussis growth in vitro and in vivo.